The Team Northern Alberta Tar Sands * Images courtesy of Syncrude - - PowerPoint PPT Presentation

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The Team Northern Alberta Tar Sands * Images courtesy of Syncrude - - PowerPoint PPT Presentation

Dec 29, 2022 108 likes •485 views

The Team Northern Alberta Tar Sands * Images courtesy of Syncrude Inc. and Shell Scotford Northern Alberta Tailings Ponds *Image courtesy of Syncrude Inc . Analogy Motility The Bacuum Cleaner Locomotion Regulation

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SLIDE 1
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SLIDE 2

The Team

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SLIDE 3

Northern Alberta Tar Sands

* Images courtesy of Syncrude Inc. and Shell Scotford

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SLIDE 4

Northern Alberta Tailings Ponds

*Image courtesy of Syncrude Inc.

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SLIDE 5

Analogy

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Motility

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The “Bacuum” Cleaner

  • Locomotion
  • Regulation
  • Degradation
  • Identification
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SLIDE 8

Overview

  • Locomotion
  • Regulation
  • Degradation
  • Identification
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SLIDE 9

Chemotaxis

Running

Amino Acids Carbohydrates

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SLIDE 10

Chemotaxis

Amino Acids Carbohydrates

Running

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SLIDE 11

Chemotaxis

P

Tumbling

Amino Acids Carbohydrates

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SLIDE 12

Chemotaxis

Amino Acids Carbohydrates

P

Tumbling

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SLIDE 13

Chemotaxis

Amino Acids Carbohydrates

P

Tumbling

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SLIDE 14

Overriding chemotaxis

Amino Acids Carbohydrates

P

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SLIDE 15

Controlling CheZ expression

2000 4000 6000 8000 4 8 12

CheZ molecules per cell Protein (M)

CheY CheY•P

Bray, D., Levin, M. D., and Lipkow, K. 2007. Curr. Biol. 17, 12-19.

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SLIDE 16

Controlling CheZ expression

2000 4000 6000 8000 4 8 12 0.0 0.2 0.4 0.6 0.8 1.0

CheZ molecules per cell Protein (M) Motility bias Motility CheY CheY•P

Bray, D., Levin, M. D., and Lipkow, K. 2007. Curr. Biol. 17, 12-19.

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SLIDE 17

Motility assay

Motile

  • E. coli WT

Non-motile

  • E. coli D cheZ
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SLIDE 18

Biobrick parts

  • cheZ
  • BBa_K147000

Promoter + control + cheZ + T1 + T2

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SLIDE 19
  • Locomotion
  • Regulation
  • Degradation
  • Identification

Overview

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Riboswitch

  • UTR of mRNA
  • Short (can be <100 nt)
  • Binds target molecule , conformational change
  • Regulates transcription termination, translation

initiation or alternative splicing

  • Fast response time
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SLIDE 21

Types of riboswitches

  • Widely diverse in

nature

  • Vary in ligands

bound, methods of regulation

  • Bacteria, archaea,

eukarya

  • Still a growing field of

research

Winkler and Breaker. 2003. Chem. Biochem. 4, 1024-1032.

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SLIDE 22

Riboswitch

  • Theophylline aptamer

+

  • Topp, S. and Gallivan, J. 2007. J. Am. Chem. Soc. 129, 6807-6811.

CheY CheZ CheY-P

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SLIDE 23

Biobrick parts

Low [ligand] High [ligand]

Promoter + RS + gene + T1 + T2

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SLIDE 24
  • Locomotion
  • Regulation
  • Degradation
  • Identification

Overview

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SLIDE 25

2-chlorobenzoate

  • Elimination of chloro substituents
  • Potential step in PCB degradation

2-chlorobenzoate (ortho-chlorobenzoate) catechol 2-hydroxymuconic semialdehyde (coloured!)

  • hb
  • peron

xylE

  • P. aeruginosa
  • P. putida

1Ingram et al. 1989. J. Bacteriol. 171, 6617-6624. 2Tsoi et al. 1999. Appl. Environ. Microbiol. 65, 2151-2161.

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SLIDE 26

Biobrick parts

  • ohb operon (ohbA, ohbB, ohbC, ohbR)
  • BBa_K147002 (ohbA)
  • xylE
  • BBa_K147003

Promoter + RS + ohb + T1 + T2 + RBS + xylE + T1 + T2

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SLIDE 27
  • Locomotion
  • Regulation
  • Degradation
  • Identification

Overview

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Identification

  • Continually monitor modified bacterium
  • Problem with reporter genes?
  • Expression limited under stressed conditions
  • Solution?
  • Translation of essential genes
  • Initiated by unique sequence
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rpsA TIR

  • Initiates translation of S1 ribosomal protein
  • Lacks Shine-Dalgarno and requirement of S1
  • Unique watermark

Hellen, C. U. T. 2007. Structure. 15, 4-6.

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Biobrick parts

  • rspA TIR

Promoter + rspA TIR + GFP + T1 + T2

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SLIDE 31

Conclusions

Locomotion Regulation Degradation Identification

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Progress

  • Modeling of CheZ function!
  • Motility assay of WT vs. DcheZ (RP1616)
  • Submitted Biobricks cheZ, xylE, ohbA
  • Characterized cheZ part
  • Isolated rpsA TIR
  • Constructed theophylline riboswitch in Biobrick

format (Nov. 6!)

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SLIDE 33

Future work

  • Continue with construct formation
  • Modeling of reprogrammed bacuum motility
  • Generate novel aptamer using SELEX
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SELEX

Systematic Evolution of Ligands through Exponential Enrichment Tetracycline selection Random nucleic acid synthesis Counter- selection with Ni2+/ Optimization Nucleic acid with selected binding

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Future work (cont’d)

  • Characterize novel riboswitch function
  • Assemble “Bacuum” Cleaner
  • Test the system!
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Acknowledgements

  • Dr. Wieden, Dr. Selinger
  • Gallivan Lab, Emory University
  • Tiedje Lab, University of Michigan